Abstract

Germline mutations of the CHEK2 gene have been reported in some myeloid and lymphoid malignancies, but their impact on development of essential thrombocythemia has not been studied. In 16 out of 106 (15.1%) consecutive patients, newly diagnosed with essential thrombocythemia, we found one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2+1G>A or del5395. They were associated with the increased risk of disease (OR=3.8; P=0.002). The median age at ET diagnosis among CHEK2+/JAK2V617F+ patients was seven years lower than that among CHEK2−/JAK2V617F+ (52 vs. 59 years; P=0.04), whereas there was no difference in the medians of hematologic parameters between these groups. The results obtained suggest that CHEK2 mutations could potentially contribute to the susceptibility to essential thrombocythemia. The germline inactivation of CHEK2, as it seems, has no direct impact on the development of disease, but it could cause disruption of cell cycle checkpoints and initiate or support the cancerogenic process of essential thrombocythemia at a younger age.

Introduction

The genetic background of essential thrombocythemia (ET) is not yet well known. Sporadic cases of ET are those most frequently observed, but patients with ET family history have also been reported, suggesting a hereditary nature of the disease.1,2 Various reports on the association between acquired and inherited gene mutations and risk of ET have been presented, but the impact of constitutional mutations of tumor susceptibility genes, e.g. CHEK2, on the disease development has not been studied to date.

CHEK2 is the tumor suppressor gene that protects cells against too rapid, uncontrolled growth by regulating of cell division. It encodes cell cycle G2 checkpoint kinase, which is a key mediator in cellular response to various types of DNA damage. The CHEK2 kinase is activated on ATM-dependent pathways and then phosphorylates substrates, such as p53, BRCA1, CDC25A and CDC25C, involved in the cell cycle checkpoint control through coordination of DNA repair, cell cycle progression and apoptosis.3

Germline CHEK2 mutations (i.e. 1100delC, I157T) were found in Li-Fraumeni syndrome,4 some lymphoid malignancies,5–7 and also in myeloid malignancies, e.g. myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), with a low frequency.6,8 Collado et al.9 described lack of CHEK2 germline mutations in Spanish patients with AML. However, the author noted that association of these mutations with cancer risk depends mainly on their incidence in the general population. Lack of a mutation, or its very low frequency in the control and study groups, make the presence of this mutation clinically irrelevant.9

Constitutional mutations of CHEK2 were reported in many types of solid tumors, including breast, ovary, colon, prostate and thyroid gland cancers. It was estimated that CHEK2 mutations cause low to moderate genetic risk of the development of solid tumors.10–14

In the general Polish population, the most common are the I157T missense mutation (4.8% frequency) and three premature protein-truncating mutations: IVS2+1G>A, del5395 and 1100delC (0.4%, 0.4% and 0.2% frequency, respectively).15 The splice-site mutation IVS2+1G>A results from nucleotide transition G→A and 4-bp insertion in the mutant transcript, due to an abnormal splicing between exons 2 and 3. The 5,395-bp deletion includes exon 9 and 10.16

In this report, we present the results of our research on association between the above mentioned four CHEK2 mutations and a risk of ET. The relationship between a congenital CHEK2 mutation and the acquired V617F mutation of the Janus tyrosine kinase 2 gene (JAK2), the important marker of myeloproliferative neoplasms,17 was also investigated, as well as its relation to patients’ age, hematologic features at ET diagnosis, as well as family history of cancer.

Design and Methods

Patients

A total of 106 consecutive patients, newly diagnosed with ET according to WHO 2008 criteria18 in two Polish hematology centers (Wrocław and Toruǹ) were involved in the study. The median age at ET diagnosis was 56.5 years (≈57 years; range 20–85). Data from medical records were collected for each patient: hemoglobin and hematocrit level, white blood cell (WBC) count and platelet (PLT) count at ET diagnosis.

In families with suspicion of a congenital CHEK2 mutation, molecular tests were performed (8 out of 16 invited families agreed to testing; a total of 22 subjects).

Fifty-nine percent of ET patients originated from families with at least one case of cancer diagnosed in a close relative. The most frequent were breast, lung, colon, stomach, pancreas, prostate and ovary cancer, as well as acute or chronic leukemia (in 6 families) or ET (in 4 families). One family fulfilled Li-Fraumeni-like syndrome criteria.19 In families of 4 patients (diagnosed with ET at the ages of 34, 62, 61 and 62 years) another ET case was diagnosed in the daughter at the age of 14, the mother at the age of 53, the sister at the age of 56, and the brother at the age of 60, respectively. Forty-three out of 106 (41%) ET patients originated from families with no other cancer case.

Medical records confirmed the ET diagnosis and the clinical history of all patients. The control group consisted of 200 healthy persons from the Wrocław and Toruǹ regions. The data on the general Polish population published by Cybulski et al.15 were used as the second control group. Informed consent was obtained from all study subjects. The study was approved by the Ethics Committee of the Collegium Medicum, Nicolaus Copernicus University in Bydgoszcz, Poland.

Molecular analysis

The CHEK2 mutations were investigated in DNA from peripheral blood leukocytes, extracted by standard salting-out method. The constitutional character of mutations was verified in mutation-positive patients by analysis of DNA from buccal swabs, extracted using Swab-Extract DNA Purification Kit (EURx, Poland). The I157T and IVS2+1G>A were examined by RFLP-PCR,20 the 1100delC by ASO-PCR,20 and the del5395 by multiplex-PCR with two primer pairs flanking breakpoint sites in introns 8 and 10.15 Mutation-positive cases were confirmed by sequencing analysis using ABI-PRISM 3130 Genetic Analyzer (Applied Biosystems, USA). The JAK2 V617F was detected in DNA from peripheral blood and from buccal swabs by allele-specific PCR described by Jones et al.,21 and cases with suspicion of homozygosity were examined by sequencing analysis.

Statistical analysis

Statistical analysis included a comparison of the prevalence of variant alleles in the studied and control groups, calculation of odds ratios (ORs) from two-by-two tables, and calculation of statistical significance of differences between tested groups using the Fisher’s exact test. Moreover, values of median and arithmetic mean (and standard deviation) of the age and hematologic parameters at ET diagnosis in groups of patients were calculated and compared. All calculations were performed using the Interactive Statistical Calculation Pages program for Windows.

Results and Discussion

Analysis of CHEK2 mutations

A CHEK2 mutation was found in 16 out of 106 ET patients (15.1%). All mutations detected in DNA from peripheral blood were also found in DNA from buccal swabs; this confirmed their constitutional character. They were significantly more frequent in the group of ET patients than in both control groups and were associated with the increased ET risk (our controls: 4.5%; OR=3.8; P=0.002; the general Polish population 5.8%; OR=2.9; P=0.0005). The elevated risk of ET also was shown in subgroups of patients: with IVS2+1G>A only, with protein-truncating mutations together, and with I157T only (Table 1). The ET risk for carriers of congenital CHEK2 mutations was comparable with their risk of breast or prostate cancer.12,15

Among patients with ET diagnosed under the age of 57 (the median age at ET diagnosis), the prevalence of CHEK2 mutations (except IVS2+1G>A) was significantly higher than among patients diagnosed with ET at/above this age compared to both controls. The IVS2+1G>A frequency was statistically higher in the group of ET patients with ET diagnosed at an older age compared to the general Polish population (Table 1).

The I157T mutation occurs in exon 3 within the highly conserved protein-interaction domain FHA (forkhead homology-associated), basic for the CHEK2 activation after DNA damage. As functional studies revealed, CHEK2 protein with the I157T alteration can not bind to and phosphorylate the downstream targets, CDC25A and p53, in phosphorylation cascades of the cell cycle. This might disrupt the regulatory function of the protein thus contributing to transformation and uncontrolled proliferation of affected cells.3

In our analysis, three frameshift mutations resulting in premature protein-truncation also led to instability of the protein and a decrease or loss of CHEK2 activity. The IVS2+1G>A eliminates part of FHA domain and the entire kinase activation domain of CHEK2 protein, fundamental for interactions with other proteins in response to DNA damage. Similarly, 1100delC and del5395 mutations result in elimination of the kinase activation domain.7,16,22 In our study, a sequence analysis of all CHEK2 mutation-positive cases confirmed their heterozygous state. Heterozygosity of mutant CHEK2 gene may cause a decrease in the CHEK2 protein concentration below the critical level and may lead to a reduction or loss of gene activity.3

The median age at ET diagnosis in 16 CHEK2 mutation carriers was four years lower than the median calculated for non-carriers, but this difference was not statistically significant (53 vs. 57 years; P=0.14). We also showed no correlation of CHEK2 mutations with any hematologic parameters, the basis for a correct diagnosis of ET (Table 2).

The investigation of 8 families confirmed the hereditary character of mutations found in ET patients by their detection in first and second degree relatives (Figure 1A-H). Out of 16 patients with a CHEK2 mutation, 7 (44%) originated from families with organ cancers (Figure 1A-D). In the family with LFL syndrome, I157T was found in the ET patient, in his healthy mother and her sister’s healthy son (a sister died from breast cancer which had been diagnosed at the age of 37). However, in the patient’s healthy father, and in a brother with childhood ALL, the I157T was not found (Figure 1C). However, among the four families with 2 cases of ET a hereditary CHEK2 mutation was shown in only one. In this family, I157T was found in 3 subjects from three successive generations, among whom 2 were diagnosed with ET (Figure 1D). Nine (21%) of CHEK2-positive ET patients originated from families with no other cancer case (4 of the families are presented in Figure 1E-H). These results suggest that CHEK2 mutations are rather associated with sporadic ET than with the familial form, although to confirm this observation a larger group of sporadic and familial ET cases should be tested.

Analysis of the JAK2 V617F

The V617F mutation was found in 61 (58%) patients, one of whom was homozygous. However, in the group of patients with ET diagnosed under the age of 57, the frequency of V617F was much lower (49%) than in the group with ET diagnosed at or above this age (66%) (Table 2). The V617F was not found in DNA from buccal swabs of ET patients, in DNA from peripheral blood of our controls, and in all but one of patients’ relatives (from 8 families). V617F was found in the daughter (diagnosed with ET at the age of 14) of the woman (diagnosed with ET at the age of 34) (Figure 1D). The homozygosity of V617F was found in a patient with CHEK2 I157T (age at ET onset 50 years) whose family did not consent to molecular studies (Table 2).

In the group of ET patients with V617F compared to the group without it, median hemoglobin and hematocrit level, as well as the WBC count at disease diagnosis were significantly higher, whereas median of the PLT count was significantly lower, findings confirmed by many authors. Moreover, the median age at ET diagnosis among V617F positive patients was five years higher than among V617F negative patients, but this difference was not statistically important (54 vs. 59 years; P=0.19) (Table 2).

Correlation of CHEK2 mutations (CHEK2+) with the JAK2 V617F (JAK2+)

The acquired JAK2 V617F mutation was found in 9 of 16 (56%) ET patients, carriers of congenital CHEK2 mutation. In 6 of 9 (67%) CHEK2+/JAK2+ patients, ET was diagnosed under the age of 57.

Medians of hematologic parameters at ET diagnosis in the groups of CHEK2+/JAK2+ and CHEK2−/JAK2+ patients were similar. However, the median age at ET diagnosis among CHEK2+/JAK2+ patients was seven years lower than among CHEK2−/JAK2+ patients (52 vs. 59 years; P=0.04) (Table 2).

These results suggest that in hereditarily predisposed individuals, with a dysfunction of the tumor suppressor CHEK2 gene which performs a key role in regulation of the cell cycle, easier and faster acquisition of JAK2 V617F mutation may occur, leading to clinical consequences at a younger age. However, it should be noted that the V617F was found in only 9 of 16 ET patients with a CHEK2 mutation. Therefore, further studies should be carried out to verify whether in CHEK2+/JAK2V617F− patients other mutations of JAK2 are present. They should also include an analysis of JAK2 46/1 haplotype, which increases the risk of ET regardless of the acquisition of V617F,23 and its relation with CHEK2 mutations.

On the basis of the results obtained, we can conclude that CHEK2 gene mutations could potentially contribute to the susceptibility to ET. It seems that the germline inactivation of CHEK2 has no direct impact on the development of ET, but it can cause disruption of cell cycle checkpoints and initiate or support the ET cancerogenic process at a younger age.

Footnotes

Funding: this work was supported by a grant from the Polish Ministry of Science and Higher Education (N402 086 32/2962).

Authorship and Disclosures

The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.

Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.